AOAC Official Method 2012.09 C.
Apparatus
Vitamins A and E in Infant Formula
and Adult/Pediatric Nutritional Formula
HPLC with UV and Fluorescence Detection
First Action 2012
A. Scope
The method involves the quantification of vitamin A palmitate,
vitamin A acetate, vitamin E acetate, and -tocopherol in infant
formula and adult/pediatric nutritional formula samples. All-trans
vitamin A palmitate and all-trans vitamin A acetate, like the free
alcohol, are susceptible to cis-trans isomerization. The 13-cis
isomer is the most common cis isomer and has 75% of the
biological potency of the all-trans isomer. One microgram all-trans
vitamin A palmitate is equivalent to 1.817 International Units (IU).
One microgram vitamin A acetate is equivalent to 2.904 IU, and
1 g free alcohol is equivalent to 3.33 IU. Similarly 1 mg dl--
tocopheryl acetate is equal to 1 IU vitamin E. One milligram d--
tocopheryl acetate, which is more active, is equal to 1.36 IU. One
milligram d--tocopherol is equivalent to 1.49 IU.
The concentration of trans vitamin A palmitate, trans vitamin A
acetate, vitamin E acetate, and -tocopherol are calculated by the
comparison of peak areas of samples to peak areas of standards of
known concentration. Quantitation of 13-cis vitamin A, the major
cis isomer of vitamin A, is accomplished by using a response factor
relative to that for the all-trans isomer. Results of the cis vitamin
A determination are added to the all-trans vitamin A results to give
total vitamin A results.
B. Principle
Samples are initially mixed with methanol, which precipitates
proteins and disrupts micelles freeing lipids for extraction. Samples
are then extracted with isooctane and centrifuged to separate the
isooctane layer from the alcohol water layer. A 20 L aliquot of the
upper, isooctane layer is injected onto the HPLC system.
The HPLC system is comprised of two, mid-bore (3.0 mm
id) silica columns placed in series and separated by a switching
valve. Following sample injection, vitamins A palmitate, vitamin
A acetate, and vitamin E acetate are separated from -tocopherol
and other extraneous compounds present in sample extracts with
a 3 cm 3.0 mm id column and a 1% methylene chloride0.06%
isopropanol in isooctane mobile phase. After vitamin A palmitate,
vitamin A acetate, and vitamin E acetate elute from the 3.0 cm
column onto a 20 cm 3.0 mm id column, a column switch takes
place and vitamin A palmitate, vitamin A acetate, and vitamin E
acetate are further separated on a 20 cm column and detected by
UV at 325 nm (vitamin A palmitate and vitamin A acetate) and 285
nm (vitamin E acetate). -Tocopherol is further separated from
other extraneous compounds on the 3 cm column and detected by
fluorescence at excitation and emission wavelengths of 295 and
330 nm, respectively.
The concentration of trans vitamin A palmitate, trans vitamin A
acetate, vitamin E acetate, and -tocopherol are calculated by the
comparison of peak areas of samples to peak areas of standards of
known concentration. Quantitation of 13-cis vitamin A, the major
cis isomer of vitamin A, is accomplished by using a response factor
relative to that for the all-trans isomer. Results of the cis vitamin
A determination are added to the all-trans vitamin A results to give
total vitamin A results.
Common laboratory equipment and, in particular, the following:
(a) Analytical balance.Capable of weighing to the nearest
1 mg, with a readability of 0.1 mg.
(b) Volumetric flasks.Class A; various sizes.
(c) Volumetric pipets.Class A; various sizes.
(d) Pipettor.15 mL variable volume with tips. Repipet
heads, 5 and 25 mL, or equivalent.
(e) Stir plate.Multiposition with appropriate size test tube
rack.
(f) Light shields.Clear UV shields with a minimum UV cutoff
of 385 nm.
(g) Centrifuge tubes.Glass, 50 mL, fitted with PTFE-lined
screw cap.
(h) Vortexer.Transfer pipets, glass. Measuring pipets, glass,
reusable, Class A, 2 mL.
(i) Centrifuge.Equipped with adapters for 50 mL centrifuge
tubes.
(j) HPLC system.One isocratic pump, one programmable
pump with flow rates ranging from 0.0 to 0.8 mL/min, one
six-port switching valve, one HPLC autosampler capable of 20 L
injections, one multichannel UV detector (vitamin A, 325 nm;
and vitamin E, 285 nm), a fluorescence detector (-tocopherol,
excitation 295 nm; emission 330 nm), and data collection system.
(k) HPLC columns.ES Industries Chromegasphere Si-60,
3 m, 3.0 30 mm, Part No. 183111 and Si-60, 3 m, 3.0 200 mm,
Part No. 143111 or Thermo Scientific BETASIL Silica-100, 3 m,
3.0 30 mm, Part No. 70003-033030 and BETASIL Silica-100,
3 m, 3.0 200 mm, Part No. 70003-203030, or equivalent.
D. Chemicals and Reagents
Use only reagents of recognized analytical grade, unless
otherwise specified, and distilled or demineralized water or water
of equivalent purity.
(a) Vitamin A acetate standard.USP retinyl acetate, official lot
with specified potency, or equivalent. Store per label instructions.
(b) Vitamin A palmitate standard.USP retinyl palmitate,
official lot with specified potency, or equivalent. Store per label
instructions. If potency information is not available it can be
determined experimentally. See Appendix A.
(c) Vitamin E acetate standard.USP all-rac--tocopheryl
acetate, official lot with specified purity or equivalent. Store per
label instructions.
(d) -Tocopherol standard.USP -tocopherol, official lot with
specified purity or equivalent. Store per label instructions.
(e) Methanol.HPLC (distilled-in-glass) grade.
(f) Methylene chloride (dichloromethane).HPLC (distilled-
in-glass) grade.
(g) Isopropanol.HPLC (distilled-in-glass) grade.
(h) 2,2,4-Trimethylpentane (isooctane).HPLC (distilled-in-
glass) grade.
(i) Reagent alcohol.
(j) Coconut oil.Food grade.
E. Preparation of Reagents and Standard Solutions
All sources of contamination must be eliminated. This includes
reagents, glassware, and all plastics except fluorinated polymers
such as Teflon, which do not contain plasticizers. Also, traces
of water, except that specifically required during the sample
preparation, must be avoided. Solutions should be prepared fresh or
whenever indications of contamination, chemical degradation, or
2012 AOAC INTERNATIONAL
 give a concentration of 1 mg/mL directly into a 100 mL volumetric
Pump 1 Autosampler, flask. If using UPS standard, weigh approximately 0.10000 g
20 uL injection
0.6-1.0
mL/min
(10%) of -tocopherol. Dilute to volume with isooctane. Store
frozen (<20C) protected from light. Expiration: 6 months.
Column 1 (f) Mixed vitamin intermediate standard.Pipet 1.0 mL vitamin
Chromegasphere Si-60,
3u, 3 x 30 mm
A palmitate stock standard solution, 10.0 mL vitamin A acetate stock
standard solution, and 25.0 mL vitamin E acetate stock standard
1 Column 2
solution into a 250 mL volumetric flask containing approximately
Chromegasphere, Si-
Fluorescence Detector
Ex: 295mm, Em: 330 nm
6 60,
0.25 g coconut oil. Dissolve and dilute to volume with isooctane.
3u, 3x 200 mm
2 Store frozen (<20C) protected from light. Expiration: 6 months.
(g) -Tocopherol intermediate standard.Pipet 20.0 mL
5
-tocopherol stock standard solution into a 100 mL volumetric
3
Pump 2 flask. Dilute to volume with isooctane. Store frozen (<20C)
4 0.6 mL/min
protected from light. Expiration: 6 months.
(h) Mixed vitamin working standards.Transfer about 20 mL
UV Detector
Vit A: 313 nm mixed vitamin intermediate standard and 15 mL -tocopherol
Vit E: 280 nm
intermediate standard into separate 25 mL volumetric flasks.
Stopper and allow to come to room temperature. Store prepared
Figure 2012.09A. System configuration 1. Valve position at working standards frozen (<20C) protected from light.
beginning of an injection. Expiration: 2 months.
(1) High working standard.Pipet 10.0 mL of mixed vitamin
intermediate standard and 5.0 mL -tocopherol intermediate
changes in concentrations are evident. Expiration dates and storage
standard into a 50 mL volumetric flask containing about 0.50 g
conditions are stated. All solutions listed below may be scaled
coconut oil. Dissolve and dilute to volume with isooctane.
up or down for convenience provided good laboratory practices
(2) Middle working standard.Pipet 4.0 mL of mixed vitamin
are observed. Preparation should be performed under shielded
intermediate standard and 3.0 mL -tocopherol intermediate
fluorescent lighting with a minimum UV cutoff of 385 nm. Avoid
standard into a 50 mL volumetric flask containing about 0.50 g
direct and indirect white lighting, especially that from ultraviolet
coconut oil. Dissolve and dilute to volume with isooctane.
lights, sunlight, and fluorescent white lights.
(3) Low working standard.Pipet 2.0 mL of mixed vitamin
(a) Mobile phase: 98.94% isooctane, 1% dichloromethane,
intermediate standard and 1.0 mL -tocopherol intermediate
and 0.06% isopropanol.Pipet 20.0 mL dichloromethane and 1.2
standard into a 50 mL volumetric flask containing about 0.5 g
mL isopropanol to a 2 L flask and dilute to volume with isooctane.
coconut oil. Dissolve and dilute to volume with isooctane.
Expiration: 2 days.
(4) Very low working standard.Pipet 1.0 mL of mixed vitamin
(b) Vitamin A palmitate stock standard solution (approximately
intermediate standard and 0.5 mL -tocopherol intermediate
2900 IU/mL).Accurately weigh an appropriate amount of
standard into a 100 mL volumetric flask containing about 1.0 g
vitamin A palmitate standard to give a stock standard concentration
coconut oil. Dissolve and dilute to volume with isooctane.
of approximately 2900 IU/mL (10%) directly into a 100 mL
volumetric flask. If using USP standard, weigh approximately
0.17000 g (10%) of standard. Add approximately 0.10 g (10%)
of coconut oil (as a stabilizer/preservative for vitamin A). Dissolve Pump 1 Autosampler,
0.6-1.0 20 uL injection
and dilute to volume with isooctane. Store frozen (<20C) mL/min

protected from light. Expiration: 6 months.

(c) Vitamin A acetate stock standard solution (approximately Column 1
Chromegasphere Si-60,
600 IU/mL).Accurately weigh an appropriate amount of vitamin 3u, 3 x 30 mm
A acetate standard to give a stock standard concentration of
approximately 600 IU/mL directly into a 100 mL volumetric flask. 1 Column 2
Fluorescence Detector
Chromegasphere, Si-60,
If using UPS standard, weigh approximately 0.72000 g (10%) Ex: 295mm, Em: 330 nm 6 3u, 3x 200 mm
of vitamin A acetate dissolved in oil. Add about 0.10 g (10%) of 2
coconut oil (as a stabilizer/ preservative for vitamin A). Dissolve
5
and dilute to volume with isooctane. Store frozen (<20C)
protected from light. Expiration: 6 months. 3
Pump 2
(d) Vitamin E acetate stock standard solution (approximately 4 0.6 mL/min

1 mg/mL).Accurately weigh an appropriate amount of vitamin E

acetate standard to give a stock standard concentration of 1 mg/ UV Detector
Vit A: 313 nm
mL directly into a 100 mL volumetric flask. If using UPS standard, Vit E: 280 nm
weigh approximately 0.10000 g (10%) of vitamin E acetate.
Dissolve and dilute to volume with isooctane. Store frozen
(< 20C) protected from light. Expiration: 6 months.
Figure 2012.09B. System configuration 2. Valve position
(e) -Tocopherol stock standard solution (approximately 1 mg/
several minutes after an injection and during test injections.
mL).Accurately weigh an appropriate amount of -tocopherol to

2012 AOAC INTERNATIONAL


Figure 2012.09C. Example test injection.
F. Preparation of Test Sample
Infant, pediatric, and adult nutritionals.Mix well to ensure
that sample is homogeneous. Powdered products that are not
homogeneous at the subgram level should be reconstituted first and
sampled immediately after the powder is completely dissolved.
Sample sizes are chosen so that after dilution with laboratory
water, if any, a total sample weight of about 4 g at a total solids value
of <20% and a maximum oil content of about 150 mg is achieved
while maintaining sample preparation concentrations of vitamin A
and vitamin E within the standard concentrations. Typically sample
sizes are 4.0 g for ready-to-feed infant formulas and 2.0 g (plus
2.0 mL water) for concentrated infant formulas. Powdered infant
formulas may first be reconstituted and treated as a ready-to-feed
infant formula or approximately 0.5 g of the powdered formula
may be weighed directly into a 50 mL centrifuge tube and diluted
with 3.5 mL laboratory water.
G. Procedure
(a) Test portion.Weigh an appropriate amount of sample
directly into a 50 mL centrifuge tube and if necessary add enough
laboratory water so that the combined weight of the sample and
any water is approximately 4 g. Water should be added rapidly to
powder samples and the sample should be immediately mixed until
all of the powder is dissolved. To each sample, add 25 mL (10%)
methanol using a re-pipet head or equivalent glassware, cap the
centrifuge tube, and vortex immediately and rapidly for at least
30 s or add a stir bar, 25 mL (10%) methanol, and begin stirring
at a rate that is fast enough to form a vortex, but does not cause
sample to splash out of the centrifuge tube. Methanol should not be
added to more than two samples, consecutively, without vortexing
or stirring.
After the addition of methanol, let samples stand for at least
10 min but not more than 40 min if vortexing or let samples stir
for at least 10 min if using a multiposition stir plate. Using a
volumetric pipet, add 10.0 mL isooctane to each centrifuge tube.
Cap the centrifuge tubes and vortex rapidly for at least 45 s or cap
the centrifuge tubes and stir at a rate that is fast enough to form
a vortex at least 2 min. Isooctane can be added to any number of
samples, consecutively, prior to vortexing or stirring.
Add 5 mL (10%) laboratory water to each tube. Cap the
centrifuge tubes and vortex rapidly for at least 20 s or stir at a
rate that is fast enough to form a vortex for at least 1 min. Water
can be added to any number of samples, consecutively, prior to
vortexing. If samples cannot be effectively vortexed or stirred, they
can be shaken by hand for at least 20 s. A sample must be repeated
from the beginning of the procedure if spillage or leakage occurs
anytime during these steps.
Centrifuge samples until there is a clean phase separation
between the isooctane and the watermethanol phase. Using a
glass transfer pipet, transfer about 1 mL of the clear upper layer
into appropriate autosampler vials.
H. HPLC Conditions
(a) System configuration 1.See Figure 2012.09A.
(b) System configuration 2.See Figure 2012.09B.
(c) System settings.
(1) Run time.2530 min depending on columns used.
(2) Pump 1 flow rate.ES Industries columns: 0.4 mL/min
011 min, 0.8 mL/min 11.129 min, 0.4 mL/min 2930.0 min.
Thermo Fisher Scientific columns: 0.3 mL/min 010 min,
0.6 mL/min 10.125 min, 0.3 mL/min 2526 min.
(3) Pump 2 flow rate.0.6 mL/min.
(4) Injection volume.20 L.
(5) Multichannel UV detector settings.Channel 1, 325 nm;
Channel 2, 285 nm.
(6) Fluorescence detector settings.Excitation, 295 nm;
emission, 330 nm.
(d) System startup.At system startup, turn on the pumps and
detector. Allow mobile phase to pass through each column for at
least h. After the system has equilibrated, auto-zero all detectors
before beginning an analysis.
(e) Column switch time determination, test injection.
(1) After the system has equilibrated, the column switch time
is determined by injecting the high working standard solution
(test injection) onto the 3 30 mm column with the system in
configuration 2. In this configuration, the standard is only going
through the 3 30 mm column. See Figure 2012.09B.
(2) After determining the retention time of vitamin E acetate and
-tocopherol peaks from the 3 30 mm column, set the column
switch time to approximately halfway between when the vitamin E
peak returns to baseline and the beginning of the -tocopherol peak
(see Figure 2012.09C). The resolution (R) between -tocopherol
and vitamin E acetate should be 2.5. See Appendix B for an
example calculation.
(f) Standard, control, and sample injections.
(1) Standard and sample run times are approximately 30 min.
Typical retention times are 68 and 79 min for 13-cis and all-
trans vitamin A palmitate, respectively; 1217 and 1421 min for
13-cis and all-trans vitamin A acetate, respectively; 1625 min for
vitamin E acetate; and 68 min for-tocopherol. See Appendix C
for example chromatograms. These times may vary somewhat from
column to column or system to system, but remain fairly constant
for a given column providing care is taken in the preparation of the
mobile phase and in treatment of the column.
(2) Confirm that the column switch time is set correctly
according to the test injection. Perform three consecutive injections
Table 2012.09. Standard dilution volumes
Working standard dilution volume
Vitamin High Middle Low Very low
Vitamin A palmitate 0.125 0.312500 0.625000 2.500000
Vitamin A acetate 0.0125 0.0312500 0.0625000 0.2500000
Vitamin E acetate 0.005000 0.012500 0.025000 0.100000
-Tocopherol 0.005000 0.008333 0.025000 0.100000
2012 AOAC INTERNATIONAL
of the high working standard and confirm that peak responses have
an RSD of 2.0%.
(3) Inject the remaining working standards, a control sample,
sample preparations, and a second set of standards. The column
switch time should be checked before and after each set of standard
and sample injections or every 24 h or when the mobile phase is
changed.
I. Determination
(a) Quantitative determination.
(1) Resolution.Visually inspect each standard and sample
chromatogram and verify that the vitamin A and vitamin E peaks
are adequately resolved. Compare the vitamins A and E standard
responses to the vitamins A and E sample responses and verify that
the sample responses are within the range of the standard responses.
Calculate the resolution (R) of the 13-cis vitamin A and all-trans
vitamin A peaks. Resolution of these isomers must be >1.85. See
Appendix B for example calculations.
(2) Linearity.The linear correlation coefficients, r, for all
standard curves must be 0.9996 and the difference in peak
responses of the same standards injected before and after a set of
samples must be 4.0%.
J. Calculations and Expression of Results
(a) Working standard concentration calculations.The
concentrations of all-trans vitamin A palmitate, vitamin A acetate,
vitamin E acetate, and -tocopherol in each of the working
standards are calculated from the amounts of standard weighed and
the declared or determined potencies or purities of the standards
using the following formula:
C= (Wt P)/D
where C = vitamin A or E concentration in IU/L or mg/L, Wt =
weight of reference standard weighed in grams, P = standard
potency or purity in IU/mg or mg/mg, and D = dilution volume
in mL. Dilution volumes for all of the standards are listed in
Table 2012.09.
(b) Standard curves.Prepared for all-trans vitamin A
palmitate, all-trans vitamin A acetate, vitamin E acetate, and
-tocopherol by performing a linear least squares routine on
standard peak areas versus corresponding standard concentrations.
The all-trans vitamin A standard curves are used for the quantitation
of both 13-cis vitamin A and all-trans vitamin A in samples. The
linear correlation coefficients, r, for all standard curves must be
0.9996 and the difference in peak responses of the same standards
injected before and after a set of samples must be 4.0%.
(c) Vitamin A calculations.Since 13-cis vitamin A palmitate
and 13-cis vitamin A acetate standards are not available, the all-trans
vitamin A palmitate and all-trans vitamin A acetate standard curves
can also be used for the determination of 13-cis vitamin A palmitate
and 13-cis vitamin A acetate using the following correction factors.
Corrected 13-cis vitamin A peak area response =
all
13-cis vitamin A peak area response
13
where Eall and E13 are the molar absorptivity of all-trans and
13-cis vitamin A, respectively, at the wavelength employed for the
determination. This ratio has been estimated to be 1.075. B is the
biological potency of 13-cis vitamin A relative to that of all-trans
vitamin A (0.75).
(d) Sample concentration calculations.The vitamin A and
vitamin E concentrations for products can be calculated from the
following equation:
Vitamin concentration, IU/kg, IU/L, mg/kg, or mg/L =
(C 10 df d)/SS
where C = the vitamin concentration of the sample preparation
obtained from the vitamin A and E standard curves in IU/L or mg/L.
For total vitamin A, the sum of the trans vitamin A concentration
and the 13-cis vitamin A concentration are added together. SS =
Sample size in grams, 10 = sample dilution volume in mL, and df*
is the dilution factor of the reconstituted powder (if applicable):
+
=
(e) Units conversions.
(1) Vitamin A results can be converted from weights to IU or
Retinol Equivalents (RE) using the following conversion factors:
(a) All-trans retinyl acetate.1 mcg All-trans retinyl acetate is
equivalent to 2.904 IU or 0.871 RE.
(b) All-trans retinyl palmitate.1 mcg All-trans retinyl
palmitate is equivalent to 1.817 IU or 0.545 RE.
(2) Vitamin E results can be converted from weights to IU or
Tocopherol Equivalents (TE) using the following conversion
factors:
(a) All-rac -tocopheryl acetate.1 mg is equivalent to 1 IU
or 0.671 TE.
(b) RRR -tocopheryl acetate.1 mg is equivalent to 1.36 IU
or 0.913 TE.
(c) All-rac -tocopherol.1 mg is equivalent to 1.1 IU or
0.738 TE.
(d) RRR -tocopherol.1 mg is equivalent to 1.49 IU or 1.0 TE.
References: J. AOAC Int. (future issue).
AOAC SMPR 2011.003: J. AOAC Int. 95, 291(2012).
AOAC SMPR 2011.010: J. AOAC Int. (future issue).
Appendix A: Vitamin A Potency
To determine potency of the vitamin A palmitate standard:
(1) Quantitatively transfer 0.1 g retinyl palmitate to a 100 mL
volumetric flask and dissolve in and dilute to volume with hexane.
(2) Pipet 2 mL of solution prepared in step 1 into a 250 mL
volumetric flask and dilute to volume with hexane.
(3) Pipet 2 mL of the solution prepared in step 2 into a 50 mL
volumetric flask and dilute to volume with hexane.
(4) Transfer a portion of this solution into a 1 cm cell path length
cuvet and measure absorbance at 325 nm.
(5) Calculate the concentration of vitamin A in mg/g using the
following equation:
325 31250
=
where Cstd = concentration of retinyl palmitate in the vitamin A
palmitate standard in mg/g; A325 = absorbance of the solution
prepared in step 3 at 325 nm; = extinction coefficient of retinyl
palmitate in hexane at 325 nm, 975 dL/gcm; b = cell path length,
2012 AOAC INTERNATIONAL
 (A)
105.00

TRANS A - 7.188
100.00

TRANS A Acetate - 18.582

95.00

90.00

85.00

80.00

AU
75.00

70.00

65.00

29.151
60.00

5.694
6.030
55.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

(B)
67.00

66.00

VIT E - 22.761
65.00

64.00

63.00

62.00

29.151
61.00

60.00

mV
59.00

58.00

57.00

56.00

55.00

29.375
54.00

53.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

(C)
Figure B1. Resolution calcuation. 240.00

A-Tocopherol - 4.872
220.00

200.00

180.00

160.00

140.00

120.00

FU
1 cm wt = weight of standard weighed 31250 = conversion factor 100.00

80.00

for g/dL to mg/g.

60.00

40.00

3.831
2.892

6.173
20.00

(6) To convert standard potency of vitamin A palmitate from 0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

mg/g to IU/g, multiply the standard potency by 1817 IU/mg.

Minutes

Figure C1. Example chromatograms: mixed working

Appendix B: Resolution Calculation
standard injections. (A) Channel 1, UV: vitamin A palmitate
See Figure B1. and vitamin A acetate at 313 nm, (B) channel 2, UV: vitamin E
acetate at 280 nm, and (C) channel 3, fluorescence:
( 2 1)
R=2( -tocopherol at ex = 295 nm, em = 330 nm.
2 1)

where R = resolution, t1 = retention time of peak 1 in minutes, t2 = (A)

retention time of peak 2 in minutes, W1 = peak width of peak 1 in 120.00
TRANS A - 7.267

115.00

minutes, and W2 = peak width of peak 2 in minutes.

110.00

105.00

100.00

In the example, t1 = 5.65 min, t2 = 7.10 min, W1 = 0.316 min, W2 95.00

90.00
AU

= 0.379 min, and R = 4.17.

85.00
CIS A - 5.750

80.00

75.00

70.00

Appendix C: Example Chromatograms

65.00
6.734
6.324
4.632

60.00

55.00

50.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

See Figures C1C3.

Minutes

(B)
70.00
VIT E - 23.238

68.00

66.00

64.00
mV

62.00

60.00
29.151

58.00

56.00

54.00

52.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

(C)
30.00

28.00

26.00
A-Tocopherol - 4.899

24.00

22.00

20.00

18.00

16.00

14.00
FU

12.00

10.00

8.00

6.00

4.00
2.909
3.143

2.00

0.00

-2.00

-4.00

-6.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

Figure C2. Example chromatograms: SRM 1849.

(A) Channel 1, UV: vitamin A palmitate at 313 nm,
(B) channel 2, UV: vitamin E acetate at 280 nm, and
(C) channel 3, fluorescence: -tocopherol at ex = 295 nm, em
= 330 nm.

2012 AOAC INTERNATIONAL

 (A)
66.00

TRANS A Acetate - 19.141

65.00

64.00

63.00

62.00

61.00

CIS A Acetate - 15.051

60.00
AU

59.00

29.150
58.00

57.00

56.00

18.152
55.00

54.00

53.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

(B)
58.00

29.151
VIT E - 23.459
57.50

57.00

56.50

56.00
mV

55.50

55.00

54.50

54.00

53.50

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

(C)
50.00
A-Tocopherol - 4.862

45.00

40.00

35.00

30.00

25.00
FU

20.00

15.00

10.00

5.00
2.904

0.00

-5.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes

Figure C3. Example chromatograms: infant formula

powder. (A) Channel 1, UV: vitamin A acetate at 313 nm,
(B) channel 2, UV: vitamin E acetate at 280 nm, and
(C) channel 3, fluorescence: -tocopherol at ex = 295 nm, em
= 330 nm.

2012 AOAC INTERNATIONAL